Some portion of calcium in foods is bioavailable, meaning it is digested, absorbed and metabolized. This bioavailable calcium affects various developmental processes including bone formation and calcification. Vegetables are potentially important dietary sources of calcium but currently most vegetables do not contain high levels of bioavailable calcium. Enhancing the concentration of bioavailable calcium in vegetables would enhance their value in providing dietary calcium. The ability to genetically alter the calcium content of agriculturally important crops is just emerging; however, how these changes may affect the nutritional aspects of calcium bioavailability is unknown. We propose to investigate how changes in the calcium partitioning, in agriculturally important plants such as carrots and the forage crop Medicago truncatula, affect nutrient availability. In carrots, we have expressed high levels of a plant endomembrane calcium transporter that doubles the calcium content of the carrot. In Medicago truncat u/a, we have identified plants that contain similar amounts of calcium, but vary in the amount they partition into the oxalate salt. Oxalate is an "anti-nutrient" and sequesters calcium in a form that is unavailable for nutritional absorption by man. This plant will serve as a model for how the level of bloavailable calcium could be enhanced in various plant foods (e.g., spinach) through the manipulation of the sequestered form of calcium. These genetically altered plants will be assayed for alterations in nutrient content and availability by measuring calcium absorption in vitro and in vivo. We will assay how diets containing these modified plants alter calcium metabolism and what affect this has on biological functions of calcium, such as bone deposition in mice and humans. The experimental approach detailed here forms a framework for evaluating the nutritional consequences of genetically modified foods. Results from this specific study should define the nutritional impact of altering the cellular content and distribution of calcium within a plant cell.